Permeation of Roxarsone and Its Metabolites Increases Caco-2 Cell Proliferation

Advances in Biological Chemistry, 2013, 3, 389-396 ABC doi:10.4236/abc.2013.34041 Published Online August 2013 (http://www.scirp.org/journal/abc/)

Permeation of roxarsone and its metabolites increases caco-2 cell proliferation

Gladys S. Bayse1*, Latanya P. Hammonds-Odie2#, Kimberly M. Jackson1, Deidre K. Tucker1, Ward G. Kirlin3

1Department of Chemistry, Spelman College, Atlanta, USA 2Department of Biology, Spelman College, Atlanta, USA 3Department of Pharmacology and Toxicology, Morehouse School of Medicine, Atlanta, USA Email: #[email protected]

Received 23 June 2013; revised 20 July 2013; accepted 27 July 2013

Copyright © 2013 Gladys S. Bayse et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT tional health concerns. We conclude that micromolar levels of these benzenearsonates are adequate to stim- The benzenearsonate, Roxarsone, has been used since ulate Caco-2 cell proliferation. 1944 as an antimicrobial, growth-promoting poultry feed additive. USGS and EPA report that Roxarsone Keywords: Benzenearsonates; Roxarsone; (4-hydroxy-3-nitrobenzenearsonate) and metabolites, Phenylarsonates; Arsenicals; Caco-2 including AHBA (3-amino-4-hydroxybenzenearsonate), contaminate waterways at greater than 1100 tons an- nually. To assess human impact of these organic arse- 1. INTRODUCTION nic water contaminants, it was important to study their potential absorption. The human adenocarcino- The number of concentrated animal feeding operations ma cell line, Caco-2, is a model for intestinal absorp- (CAFOs) has increased in the US since the late 1980s. tion. We found proliferative effects on Caco-2 cells at Governmental agencies and public health groups have in- micromolar levels of these compounds, as monitored dicated that farming practices account for declining wa- by [3H]-thymidine incorporation into DNA. Flow cy- ter quality in the US [1,2]. From the 1940s, benzenearso- tometry cell cycle analysis confirmed accumulation in nates have been used as approved feed additives to pre- S phase from 21% (control) to 36% (24 hour expo- vent infections and to promote growth in commercially sure to 10 μM AHBA). Confluent Caco-2 cells grown grown poultry and swine [3]. Roxarsone (4-hydroxy-3- on collagen-coated Transwell plates were dosed on the nitrobenzenearsonate) is the major feed additive used for apical side. After exposure, media from apical and broiler production [4]. Roxarsone and its metabolites are basolateral sides were collected separately. Following primarily excreted, adding more than 1100 tons per year removal of FBS by 30K centrifugal filtration, the ben- to waterways, especially downstream from CAFOs [5-7]. zenearsonates in the collected media were analyzed Additionally, poultry litter (excrement, spilled feed, cage by HPLC. Analyses were at wavelengths in the ultra- straw) is frequently used as fertilizer [8]. Some states violet/visible range where the absorbance values were have recently banned or decreased poultry litter use as linear with respect to concentration. Concentrations fertilizer, but enforcement procedures are not clear [9,10]. were calculated by comparison with analytically-pre- The highest density of poultry CAFOs exists in the Del- pared commercial standards. Results from cells dosed marva Peninsula (Delaware, Maryland, and Virginia), the at 10 μM for 24 hours with AHBA, Roxarsone, or Ozark Plateau (Arkansas, Oklahoma, Missouri, and some Acetarsone indicated 6% - 29% permeation occur- areas of eastern Texas), Georgia, and the Carolinas [5]. ring from apical to basolateral side, modeling absorp- The areas downstream of poultry CAFOs where the popu- tion across intestinal epithelium to the circulatory sys- lations’ drinking water is provided by the local ground- tem. Benzenearsonate feed additives are frequently ap- water are of particular concern [11]. plied in combination with antibiotics, raising addi- The Roxarsone reduction product, AHBA (3-amino-4- hydroxybenzenearsonate), has been identified by poul- *Dr. Gladys Bayse deceased suddenly on July 25th, 2013. #Corresponding author. try excretions [12] and produced by anaerobic bacterial

Published Online August 2013 in SciRes. http://www.scirp.org/journal/abc 390 G. S. Bayse et al. / Advances in Biological Chemistry 3 (2013) 389-396 action [13]. The structural formulas for Roxarsone bio- 2. MATERIALS AND METHODS transformations to the reduced AHBA and N-acetylated 2.1. Chemicals Acetarsone are shown in Figure 1. Several studies have revealed rapid AHBA and inorganic arsenic (iAs) pro- Roxarsone and AHBA were obtained from Pfaltz and duction from Roxarsone, using both chicken litter and Bauer, Inc. (Waterbury, CT). Acetarsone, RNase A, and anaerobic microbial-containing sludge [13]. propidium iodide were obtained from Sigma-Aldrich (St. A pure Clostridium strain has been reported to reduce Louis, MO). HPLC grade water was purchased from Roxarsone to AHBA [14]. Aromatic amines such as ThermoFisher Scientific Co., Suwanee, GA. The human AHBA easily form azides that are usually toxic and often colon adenocarcinoma Caco-2 cell line, Eagle’s MEM carcinogenic [15,16]. We previously observed that the (EMEM) growth media and Fetal Bovine Serum (FBS) Roxarsone metabolite, AHBA, can be N-acetylated to were obtained from American Type Culture Collection Acetarsone (3-N-acetylamino-4-hydroxybenzenearsonate) (Manassas, VA). in vitro by cloned human N-acetyltransferase 1 [17]. The acetylation rate of AHBA to Acetarsone is slow when 2.2. Supplies compared to the model substrate, p-aminobenzoic acid, Microsep 30K centrifugal microconcentrators from Pall and to the swine feed additive, Arsanilate [18]. Acetarsone Life Sciences were purchased from VWR Scientific Co. had three times the toxicity of Roxarsone in the rodent (Suwanee, GA) and were used to remove FBS from model [19]. culture media prior to LC analysis. Centrifugation was The FDA recommendation to reduce the use of Rox- at 5˚C and 5000 × g. Transwells were a product of arsone in poultry feed may impact water contamination Corning Costar Corp. (Tewksbury, MA) and were a 6 downstream from CAFOs [20]. However, investigators well format of 0.4 micron pore size with collagen coat- have shown that years of soil penetration by Roxarsone ed PTFE membranes. The Whatman 4 mm syringe fil- and its metabolites is such that leaching into ground wa- ters with a pore size of 0.45 microns were purchased ter is likely to continue for some time [5,7]. Chronic ex- from ThermoFisher. posure of humans to such arsenic-containing compounds, especially in the water supply, is a matter of serious con- 2.3. Cell Culture for Proliferation, Cell Cycle cern. The nature of the organic moiety in feed additives Analysis and Transwell Studies is also of concern, since structurally similar aromatic com- Cells were cultured in EMEM media supplemented pounds can be metabolized to active electrophiles, poten- tially reacting with DNA and proteins to cause tissue ne- with 20% FBS, 100 U/ml penicillin G, 100 µg/ml strep- crosis and/or to form mutagens [21,22]. tomycin, 0.25 µg/ml amphotericin B, 1.0 mM sodium This laboratory has reported proliferative effects of pyruvate, and 2.0 mM L-glutamine and incubated in a these compounds both on freely cycling and on synchro- humidified 5% CO2 atmosphere at 37˚C. Cell viability nized Caco-2 cells, the human colon adenocarcinoma was determined microscopically by exclusion of Trypan cell line [17,23-26]. These observations were confirmed Blue. Prior to treatment with benzenearsonates, cells by using flow cytometry analysis after dosing the cells used in the proliferation and cell cycle analysis studies with micromolar levels of Roxarsone, AHBA, and Acet- were gradually serum deprived to zero over a four-day arsone [23]. In this report, we tested the hypothesis that period, followed by addition of 20% serum to synchro- if benzenearsonates are present in drinking water, these nize the cells to move from G0 into the proliferative compounds can be absorbed upon ingestion. Data in our phase of the cell cycle. preliminary reports indicated that concern over potential 2.4. Cell Proliferation human impact of benzenearsonates in drinking water was justified [17,23]. Thus, we postulated that it was impor- Cultures were replicated in quadruplicate on ninety-six tant to study their absorption and possible metabolism by well plates, seeded with 0.5, 1.0, and 2.0 × 104 cells, intestinal epithelial cells. The Caco-2 cell line is a model synchronized and at 70% - 80% confluence, were ex- frequently used for that purpose by the pharmaceutical posed to micromolar concentrations of Roxarsone, AHBA, industry [27-29]. Caco-2 cells are adherent and are ex- and Acetarsone for 16 and 24 hour periods. Cells were tensively used for permeability studies involving Trans- then incubated with [3H]-thymidine for 6 hours and har- well devices. In an effort to mimic absorption from the vested (Skatron Cell Harvester, Sterling, VA) onto glass intestinal lumen across the epithelial monolayer into the fiber filters. The filters were placed in scintillation vials. circulatory system, we dosed Caco-2 cell on the apical Radioactivity incorporated into DNA was determined side with the benzenearsonates, Roxarsone, AHBA and by a scintillation counter as a relative measure of cell Acetarsone. proliferation and expressed as percent of control, desig-

Copyright © 2013 SciRes. ABC G. S. Bayse et al. / Advances in Biological Chemistry 3 (2013) 389-396 391 nated as 100%. For tightness of replicates, data were 2.7. HPLC analyzed by coefficient of variation (standard deviation/ The HPLC System (ThermoSeparations Products, Ri- mean). viera Beach, FL) was a C-18 Aquasil column (Therm- oElectron, Bellefonte, PA), 4.6 × 250 mm, with a mo- 2.5. Cell Cycle Analysis by Flow Cytometry bile phase of 17 mM ammonium acetate buffer pH 6.0, Caco-2 cells were replicated in triplicate in six well a flow rate of 1 ml/min, and a variable wavelength ul- plates and seeded with 2.0 × 104 cells, synchronized traviolet/visible detector. For injected samples, Roxar- and at 70% to 80% confluence exposed to 10 M Rox- sone was detected at 290 nm or 400 nm with a retention arsone, AHBA, and Acetarsone for 16 and 24 hour time of 3.5 minutes; AHBA at 290 nm, 3.2 minutes; and periods. Cells were harvested, separated from media by Acetarsone at 245 nm, 4.5 minutes. The absorbance of centrifugation and fixed at −20˚C with 70% ethanol samples from the control wells was analyzed at the in- overnight. The cells were then pelleted by centrifuga- dicated wavelengths for each compound and subtracted tion, resuspended in 1 ml of PBS containing 1 mg/ml of from the dosed benzenearsonate absorbance values, when glucose, 1 mg/ml of RNase A and stained with 50 g/ml determining concentrations. A minimum of 4 injections of propidium iodide. A FACS Calibur equipped with from each of the apical and basolateral triplicate wells dual argon-ion lasers (Becton-Dickinson Immunocyto- from the Transwell experiments were analyzed. Con- metry Systems, San Jose, CA) was used to perform flow centrations were determined by peak height comparison cytometry and data were recorded, stored, and analyzed with the three commercial standards prepared at 10 μM. by ModFit-LT (Verity Software House) and Cell Quest software (Becton-Dickinson). 2.8. Statistical Analysis 2.6. Transwell Studies One-way ANOVA and Newman-Keuls analysis were used to determine the significance in Roxarsone perme- Cultures were grown to about 90% confluence on colla- ation over time. gen-coated membranes as determined by microscopic in- spection and measurement of electrical resistance. The 3. RESULTS cultures typically required about 14 days of culture to reach confluence. The electrical resistance was measured There are several literature reports of Roxarsone and two using “chopstick electrodes” with an Evometer (World metabolic products, the nitro-reduced AHBA arising Precision Instruments, Sarasota, FL). The blank well serv- from microbial sources [13], and the N-acetylated Acet- ed as reference for the electrodes and the desired reading arsone from animal [12] and human sources [23]. These prior to dosing was about 170 ohms (resistance readings three compounds, Roxarsone, AHBA and Acetarsone were stable over the 48 hour treatment period). The con- (Figure 1), are the focus of data presented here on pro- trol wells were not dosed, however were processed as in- liferative effects, flow cytometry analysis, and Transwell dicated below. The confluent monolayers were dosed studies, employing the Caco-2 cell culture model. The with 10 μM Roxarsone, AHBA, or Acetarsone in the com- proliferative effects of Roxarsone, AHBA, and Acetar- plete media for 12, 24, or 48 hour periods. After incuba- sone on synchronized Caco-2 cells were examined using tion, media on the apical and basolateral sides were col- two independent methods, cell proliferation monitored 3 lected into separate tubes. Then centrifugal filtration was by [ H]-thymidine uptake and cell cycle analysis using 3 used to remove FBS. Basolateral media samples were flow cytometry. [ H]-thymidine uptake into DNA as de- concentrated 5-fold and filtered through 0.45 micron ny- scribed in MATERIALS AND METHODS was used as lon filters, before injection onto the HPLC systems. The a measure of DNA synthesis primarily occurring during absorbance of samples from the control wells, at the HPLC wavelengths and retention times indicated, were subtracted from the benzenearsonate experimental sample absorbance values, when determining concentrations. Apical media samples were analyzed without concentration or filtration through 0.45 micron filters. The amounts of Roxarsone, AHBA, and Acetarsone in both basolateral and apical media were analyzed. The 10 μM dose in a 1.5 ml volume (apical) equals 15 nmoles. Three independent experiments were conducted, each of triplicate wells Figure 1. Structural formulas of roxarsone, AHBA, and Acet- dosed with Roxarsone, AHBA or Acetarsone. arsone.

Copyright © 2013 SciRes. ABC 392 G. S. Bayse et al. / Advances in Biological Chemistry 3 (2013) 389-396 the S phase of the cell cycle. Proliferation values result- presence of AHBA, there was a noticeable increase in the ing from exposure to each compound for 16 and 24 hour percentage of cells in S phase of the cell cycle correla- over the 0.2 - 10 µM dose range, expressed as percent of ting with increased cell proliferation (Figure 2). Neither control are shown (set as 100%; Figure 2). After 16 Roxarsone nor Acetarsone significantly altered the pro- hours of exposure, all three compounds resulted in slight portion of cells in these cell cycle phases compared to increases over the control. control treatments (Figure 3). The data from both the Likewise, after 24 hours, all compounds at all dose [3H]-thymidine uptake and the flow cytometry experi- levels caused increases, ranging from 111 to 164 percent ments indicated that AHBA increased the proliferation of relative to control values (Figure 2). AHBA was consis- synchronized Caco-2 cells. tently the greatest stimulator of proliferation at 24 hours To investigate the ability of Roxarsone, AHBA, and at all tested doses. Flow cytometry analysis was used Acetarsone to readily transverse a monolayer of epithe- also to evaluate the proliferative potential of Roxarsone lial cells, Transwell studies using Caco-2 cells were per- and its two metabolites. Flow cytometry data for syn- formed. The results of these Transwell studies in which chronized Caco-2 cells exposed to all three compounds Caco-2 cells were treated for 24 hours with 10 μM (15 at 10 µM for 24 hours are shown in Figure 3. In the nmol) of one of the benzenearsonates administrated to the apical side of the monolayer are shown in Table 1. For Roxarsone and Acetarsone, the apical and basolateral compartments’ amounts totaled approximately 15 nmol, which is equivalent to the original 10 μM applied dose. This implied that no net metabolic conversion of these compounds occurred in the Caco-2 cells during the 24 hour incubation period for the Transwell studies, but that these compounds readily transversed the epithelial monolayer. At 24 hours, the percent permeation from apical to basolateral sides was somewhat different between the

compounds, 25% (3.78 ± 0.19 nmol) of dosed Roxarsone Figure 2. Caco-2 cell proliferation in response to 0.2 - 10 μM and 29% (4.37 ± 0.20 nmol) of dosed Acetarsone (Table Benzenearsonates for16 and 24 hours. Left to Right: Control, Roxarsone, AHBA, Acetarsone (as indicted by bar patterns in 1). AHBA exhibits only 6% permeation (0.87 ± 0.21 legend). Bars indicate percent of control rates of proliferation, nmol) from apical to basolateral sides. The apical side re- expressed as Mean +/− SE from a sample size of n = 3 (4 rep- tained 49% of the original AHBA dose, giving a total re- licates per n). covery of 55% of the 15 nmol dose (Table 1). Recovery of dosed AHBA presented analytical challenges and we have been unable to account for the total dosed amounts. However, plots (not shown) of 24 and 48 hour dosing for AHBA and for Acetarsone indicated time-dependent mi- gration. The permeation by Roxarsone across the Caco-2 monolayers showed time-dependent linearity at 12, 24 and 48 hour exposure to the 15 nmol dose (Figure 4).

Table 1. Degree of permeation of Caco-2 cells by 10 μM (15 nmol) doses of Roxarsone, AHBA, and Acetarsone after 24 hours exposure.

Roxarsone Acetarsone AHBA

Basolateral

Permeation, nmol 3.78 4.37 0.87

SE 0.19 0.20 0.21 Figure 3. Flow cytometry data for Caco-2 cells dosed with Roxarsone, AHBA, and Acetarsone (10 µM, 24 hours). Caco-2 Apical cells were exposed to 10 µM benzenearsonates for 24 hours, and then processed for cell sorting according to the procedure Concentration, nmol 11.01 10.64 7.41 described in Materials and Methods. Average percentage of SE 0.35 0.35 0.58 cells (n = 3) from 3 independent experiments.

soil ultimately into groundwater [5-7]. Microbial action on Roxarsone to produce AHBA has been reported from several studies. Recent papers using anaerobic microbial-containing sludge and Clostridia strains known to be in chicken litter reveal both AHBA and iAs production from Roxarsone [13,14]. In spite of the above concerns, the July 2010 Federal Register sum- mary notice (21CFR 558.530) still gave approval for continued use of Roxarsone as a feed additive for poultry, turkeys and swine [3]. However, following additional analyses of iAs content of chicken livers reported by the FDA, the major commercial manufacturer in the United Figure 4. Permeation rate of Roxarsone (15 nmol) States, Alpharm, Inc., a subsidiary of Pfizer, voluntarily from apical to basolateral membrane of Caco-2 cells suspended sales of Roxarsone (3-Nitro) in July 2011 over 12-48 hours of exposure. Amount of Roxarsone [20]. measured on basolateral side of membrane 12, 24 and In the current studies, the proliferative effects of Rox- 48 hours after administration to the apical side in arsone and its metabolites, AHBA and Acetarsone, on Transwell studies described in Materials and Methods. One-way ANOVA indicated significant changes in Rox- Caco-2 cells were examined. While all three compounds arsone amounts between time points and Newman- resulted in increased DNA synthesis as monitored by 3 Keuls indicated significant differences between each [ H]-thymidine uptake (Figure 2), only AHBA caused a time point (p < 0.001). notable increase in the percentage of cells accumulating in S phase (Figure 3) as measured using flow cytometry. 4. DISCUSSION The potential for human health impacts of Roxarsone and its metabolites hinges on absorption of these com- This country’s food supply is regulated at the federal pounds from ingesting contaminated drinking water. For level and considered generally safe in comparison with all three benzenearsonates, permeation occurred at the 10 other countries. However, the food supply contains sev- μM dosage level (Table 1). There was a linear relation- eral thousand additives in trace amounts, including vet- ship for transport across the epithelial monolayer of erinary pharmaceuticals and feed additives, in the case of Caco-2 cells over the 12 to 48 hour treatment period for animal products. A large percent of consumable animal Roxarsone (Figure 4). The amount that permeated the protein comes from animals exposed to “medicated” feeds monolayer is equivalent to 3.78 ± 0.19 nmol (25%) for for part of their lives [30]. Most poultry farms in the Roxarsone, 4.37 ± 0.20 nmol (29%) for Acetarsone and United States have used Roxarsone as a feed additive [8]. 0.87 ± 0.21 nmol for AHBA (6%; Table 1). The total Approval was given by the FDA in 1944 for its use to amount of Roxarsone and Acetarsone detected in the treat coccidiosis and improve growth in broilers. The Na- apical and basolateral media samples from the Transwell tional Toxicology Program data raised concerns because studies approximated the dosed amounts, suggesting that of toxicity in rats (200 ppm) and mice (800 ppm) ex- there was no metabolic conversion of these benzenear- posed to Roxarsone in a thirteen week study [31] and sonates. In contrast, recovery of the dosed amount of because of positive results with Roxarsone in Salmonella AHBA was only 55%. The possible oxidation products mutagenicity tests [32]. In addition, there has been large- of AHBA to Roxarsone or potential N-acetylation to scale contamination of waterways by feed additives and Acetarsone were not detected in the media samples. metabolites due to the trend from the 1980s toward con- Stoltz et al. [14] have noted from their electronic struc- centrated animal feeding operations [16]. ture analysis that reduction of the nitro group of Rox- A major USGS paper on organic water contaminants arsone to the amine of AHBA is likely to occur before in urban areas did not contain the planned analysis of the loss of iAs from the ring. This recognizes the bond Roxarsone content due to incompatibility with the analyt- strength of the stronger aromatic carbon to arsenic (C-As) ical methods in the study [1]. Until recently, chicken litter bond versus the alkyl C-As bond. Given the chemical (excrement, spilled food, and cage straw) was widely us- reactivity of the AHBA molecule (Figure 1), even loss of ed as fertilizer on the same farms, leading to further pol- water from the arsenate moiety would destabilize the lution of soil and groundwater. Several states began to ring, enhancing its cleavage. Such a reaction could ex- decrease or monitor the use of litter as fertilizer but the plain our inability to account for all the AHBA in the extent of enforcement at the time was unclear [2,9,33]. Caco-2 Transwell studies (Table 1), since the HPLC de- Concerns were also expressed by several investigators tection of AHBA was by ultraviolet absorbance of its regarding the leaching of Roxarsone from such fertilized aromatic ring.

Unfortunately, there have been no values reported for Acetarsone. As a consequence of the current awareness levels of these compounds in drinking water for com- (FDA recommendations and the major manufacturer’s parison. The Caco-2 monolayer model mimics the poten- halt in sale of Roxarsone) of the potential for longer term tial absorption of compounds into the circulation [27-29]. impacts on human health from the Roxarsone in poultry After reaching the bloodstream, most substances travel to feed, the levels of it and its metabolites including iAs the liver. Our observed proliferative effects in human cell should decrease over time downstream from the CAFOs. cultures raise concerns regarding the possibility of harm- Unfortunately, a recommendation to reduce the use of ac- ful products from hepatic enzyme activity. Proliferation cumulated poultry manure as fertilizer or to stop the in- may not be directly linked to tumorigenesis, but increas- clusion of Roxarsone in cattle feed, has not been forth- ed proliferation could result in increased sensitivity to coming in this country. However, the years of soil per- carcinogens and/or mutagens [34]. We have observed meation will continue to cause leaching of these arsenic oxidation of NADPH with AHBA as substrate and cyto- containing compounds into drinking water for some time. chrome P450 CYP3A4 as catalyst; but no product has yet Furthermore, any enforcement of regulations and recom- been identified [23]. mendations on Roxarsone usage has been left to the states Continued study of these organoarsenical metabolites and concerns over human health impact appear not to and their biological role(s) in increasing cell proliferation have been addressed [9]. However, EPA will investigate is essential and attention should be focused on possible if the Inspector General is alerted to abuses [10]. products resulting from bioactivation. In animals, no en- There exists a growing body of evidence from the sci- zyme has been isolated and characterized for the reduc- entific community that the increase in antibiotic resistant tion reaction of Roxarsone to AHBA. However, aromatic microbes is partly attributable to the widespread use and nitro reduction is commonly attributed to liver CYPs, poorly controlled disposal of waste from animal feeding NADPH-quinone oxidoreductase, or intestinal anaerobic microbes [22]. Production of AHBA from Roxarsone by operations using arsenic-containing feed additive/antibi- poultry had been reported many years ago, [12]. Evidence otic combinations [45-47]. A survey of the Code of Fed- for anaerobic microbial conditions that produce AHBA eral Regulations list for Roxarsone reveals that its appro- from Roxarsone was provided [14]. Also, AHBA, the val for use in poultry and swine feed permits combina- most proliferative metabolite in our studies, has an aryla- tions with over twenty different antibiotics [48]. In light mine structure making it a candidate for DNA-adduct of their likely roles as drinking water contaminants with formation [35]. Possible metabolic reactions for AHBA implications for human health, the continued approval could also produce a reactive nitroge n species, as a posi- and use of other organic arsenicals as animal feed addi- tively charged nitrene or a free radical [22]. Roxarsone tives (Arsanilate in swine and poultry feed, swine waste and Acetarsone are also proliferative, but with lesser in- as fertilizer and Nitarsone and Carbasone as animal me- creases in S phase than AHBA. dicinals) need to be evaluated. Several authors have noted the dichotomy of arsenicals’ medicinal benefit versus deleterious effects on cells 5. ACKNOWLEDGEMENTS and tissues. Information is scarce on aromatic nitro and The authors would like to thank Brittney L. Thirkield for her assistance amino compounds’ specific roles in mutagenicity and carcinogenicity [32]. For example, in a study of human with manuscript preparation, Dr. Marisela De Leon Mancia for her gastric cancer MGC-803 cells, Zhang et al. [36], found technical assistance with and analysis of the flow cytometry studies, 3 that Arsacetin (4-N-acetylbenznearsonate; N-acetylarsa- Aisha Rollins-Hairston for her technical assistance with the [ H]-thymi- nilate) treatment resulted in increased protein kinase ac- dine studies, and Dr. Cornelia D. Gillyard for her insight into organic tivity, apoptosis, p53 expression, and cell proliferation mechanisms for benzenearsonates. opposite results were obtained with micromolar levels of This work was supported by grants from the National Institute of General Medical Sciences at the National Institutes of Health [Grants iAs (As2O3). Other investigators have confirmed that micromolar levels of iAs caused DNA damage, cell arrest, GM008241 and 1SC3GM083747 to G.S.B.; Grant GM008241 to and probable reduction of levels of cyclin/CDKs [37,38]. L.P.H.O. and Grant GM028248 to W.G.K.]; the National Center for iAs at micromolar levels have public health outcomes in Minority Health Disparities at the National Institutes of Health [Grant occurrence of cancers and metabolic diseases, endocrine MD000215-SP0001 to K.M.J.]; L.P.H.O. and K.M.J. were supported as disruption, and altered immune response [39-44]. Scholar-Teachers [Minority Institutions of Excellence/National Aero- The data presented here show transport of nanomole nautics and Space Association NCC8-227]. Partial support was pro- levels of benzenearsonate compounds by Caco-2 cells, vided by the CB2RSpelman College Core Laboratory which has fund- with subsequent proliferative effects. The ultimate goal ing from the National Center for Minority Health Disparities at the of this project is to elucidate the mechanism(s) that ex- National Institutes of Health [Grant MD00215]. Neither the NIH nor plain the proliferative effects of Roxarsone, AHBA, and NASA had a role in study design; in the collection, analysis and inter-

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